We propose to study the stoichiometry, thermodynamic relationships, and mechanisms by which electrochemical proton gradients are generated by electron transport in the mitochondria of tumor cells. We will measure the number of H ion ejected as electrons pass through each of the energy-conserving sites of the respiratory chain of mitochondria isolated from malignant cells using sensitive electrodes and spectrophotometry. We shall also establish the number of H ion absorbed by mitochondria during synthesis of ATP and the number of H ion ejected during ATP hydrolysis, again in the mitochondria isolated from various types of malignant cells. The relationship of this proton stoichiometry to the energy contributions of aerobic glycolysis and respiration in malignant cells will also be developed quantitatively. We also propose to study the mechanism of energy-coupled Ca2 ion influx and efflux in tumor cell mitochondria and the molecular signals that regulate Ca2 ion influx and efflux, particularly the oxidation-reduction state of mitochondrial pyridine nucleotides. The goal is to determine the role of the mitochondria in the homeostatic regulation of the concentration of Ca2 ion in the cytosol compartment. We shall examine the transport of lactic acid across the plasma membrane of intact tmor cells and its relationship to the acid-base balance in the cytosol and mitochondrial compartments of tumor cells, the intrinsic H ion permeability of the mitochondrial and plasma membranes of tumor cells, and the uptake and release of Ca2 ion by intact tumor cells. We shall employ mitochondria from Ehrlich ascites, AS30D tumor cells, L1210 cells, and the Novikoff hepatoma. For crucial tests of specificity we will use normal fibroblasts transformed in tissue culture with oncogenic viruses.